Monday, September 12, 2011

Laboratory Detection of HCV core antigen.

The introduction of a reliable and sensitive HCV core antigen assay was hampered by different difficulties: (i) the development of specific monoclonal antibodies recognizing all different HCV subtypes; and (ii) the need of accumulation and dissociation of HCV particles from immune complexes for increasing sensitivity. Recently, a first HCV antigen detection system was approved by the legal authorities and has become commercially available in the US and Europe (Ortho, trak-CTM, Ortho Clinical Diagnostics, Raritan, NJ, USA). In this assay, after dissociation of HCV particles from immune complexes and lysis, HCV core proteins are bound to coated monoclonal antibodies in a microwell. Following several washing steps a bound core antigen is incubated with an anticore-specific Fab antibody fragment conjugated with horseradish peroxidase. After a second wash quantitative detection is performed by the addition of ophenylenediamine (OPD)/hydrogen peroxide and measurement of the optical density. HCV core concentrations are calculated against a curve obtained from standards and are expressed as picograms per milliliter. The limit of detection established by the manufacturer is 1.5 pg/mL. The HCV core antigen assay is highly specific (99.5%), genotype independent, and has shown a low inter- and intra-assay variability (coefficient of variation 5–9%) (Bouvier-Alias et al., 2002 and Veillon et al., 2003).
After infection with HCV detection of HCV core antigen is delayed approx. 1–2 days after HCV-RNA becomes detectable (Icardi et al., 2001 and Cividini et al., 2003).
As for HCV-RNA, no correlation between levels of HCV core antigen and elevation of liver enzymes or the grade of inflammation and the stage of fibrosis in the liver was observed. From the results of different studies it was shown that the lower detection limit of 1.5 pg/mL for core antigen equals to approximately 10,000–50,000 IU/ml (Bouvier-Alias et al., 2002 and Veillon et al., 2003).
In a study with 139 HCV antibody and HCV-RNA positive patients presented in an outpatient clinic six (4%) were HCV core antigen negative. In these patients HCV-RNA concentrations were measured between 1,300 and 58,000 IU/mL highlighting the limitations to use the HCV core antigen assay for confirmation of ongoing hepatitis C in HCV antibody positive patients (Veillon et al., 2003).
Comparison of HCV core antigen and HCV-RNA quantification show an excellent correlation (correlation coefficient, r =0.92). One pg/mL of HCV core antigen equals to 8,000 IU/mL HCV-RNA with a intersubject variation from 5,000 to 12,000 IU/mL (Bouvier-Alias et al., 2002 and Veillon et al., 2003) Studies for evaluation of the utility of HCV core antigen concentrations for prediction of virologic response/non-response before and during interferon-based antiviral therapy are currently under way. Preliminary data of a small study investigating 38 patients treated with peginterferon-alfa and ribavirin observed a potential use of a positive HCV core antigen test (>1.5 pg/mL) for discontinuation at week 12 with a high negative prediction value (100%non-response) (Buti et al., 2004).
However, as shown by others, due to the limited sensitivity transient negative results by the HCV core antigen assay were associated with continuously positive HCV-RNA concentrations. The aim of determination of early virologic response certainly is to discontinue as many virologic non-responders as possible from further treatment with an ideal 100% prediction value. Thus, the proportion of nonresponder patients to be discontinued from further treatment at week 12 and 24 by core antigen and HCV-RNA measurement, respectively, has to be carefully assessed in large cohorts of patients (Rebucci et al., 2003).